Although magnesium is the most abundant divalent metal ion found in most cells, the regulation of the cytosolic free magnesium ion concentration (Mg-i), and the role which alterations in the level of this ion may play in mediating the pathology of a variety, of toxins, is relatively unknown. In order to attack this problem, we have previously designed and synthesized both NMR and fluorescent indicators which can be loaded into cells and are sensitive to the free magnesium ion level. Over the past year we have applied these indicators to the study of mechanisms which regulate Mg-1. In order to understand how alterations in Mg-i might be involved in cell injury, it is important to understand the normal physiological regulation of Mg-i. We varied the ionic composition of the medium in which the cells were perfused and obtained evidence for an indirect effect reflecting competition between intracellular calcium and magnesium ions for binding sites. To understand the mechanisms involved in modulating Mg-i, we examined the effect of altering cytosolic pH. Cultured embryonic chick heart cells were grown on coverslips and loaded with either the fluorescent Mg-i indicator FURAPTRA, or the pH indicator BCECF. Internal pH was altered by exposing the cells, to 10 mM NH4CL . When neutral NH3 crossed the membrane, the intracellular pH rose transiently to about 7.9; this was accompanied by a 0.18 mM decrease in Mg-i. After a few minutes, pH recovered to 7.25 and Mg-1 returned to control levels. Upon removal of extracellular NH4Cl, intracellular pH fell to 6.5, whereas Mg-i increased by only 0.06 mM. Total cell magnesium measured by atomic absorption spectroscopy was not altered, by these manipulations. Although the dissociation constant of calcium ions from FURAPTRA of 53 uM is well above basal cytosolic calcium levels, variations in intracellular calcium can be a significant limitation on Mg-i determinations using this indicator. In order to overcome this shortcoming, we have devoted a significant effort toward the synthesis of indicators with improved selectivity for magnesium/calcium.